1. Field of the Invention
The present invention relates to a method and apparatus for amplifying nucleic acids, and more particularly, to a method and apparatus for amplifying trace amounts of nucleic acids to a level necessary for a specific analysis.
2. Description of the Related Art
To disclose the genetic information of nucleic acids such as DNAs and RNAs for the purpose of sequence analysis and disease diagnosis, amplification of trace amounts of nucleic acids to a desired level is required. As nucleic acid amplification techniques well known in ordinary persons skilled in the art, there are a typical isothermal amplification technique, such as ligase chain reaction (LCR), strand-displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), and loop-mediated isothermal amplification (LAMP), and a non-isothermal amplification technique, such as polymerase chain reaction (PCR) that has been recently mainly used.
Among the amplification techniques, the PCR is performed by repeated cycles of three steps: denaturation, annealing, and extension. In the denaturation step, a double-stranded DNA is separated into two single strands by heating at 90° C. or more. In the annealing step, two primers are each bound to the complementary opposite strands at an annealing temperature of 55 to 65° C. for 30 seconds to several minutes in conventional PCR machines. In the extension step, DNA polymerase initiates extension at the ends of the hybridized primers to obtain DNA double strands. The time required for the extension step varies depending on the concentration of a template DNA, the size of an amplification fragment, and an extension temperature. In the case of using common Thermusaquaticus (Taq) polymerase, the primer extension is performed at 72° C. for 30 seconds to several minutes.
However, with respect to nucleic acid amplification according to the above-described PCR technique, when nucleic acids are present in trace amounts, PCR efficiency may be lowered. For this reason, there arises a problem in that amplification is hardly performed or nonspecific PCR products are often produced.
To solve this problem, Kemp et al. suggested a Nested PCR technique in which a two-step PCR is performed using an outer primer pair and an inner primer pair (David J. Kemp et al., 1989, Proc. Natl. Acad. Sci. Vol. 86, 2423˜2427). That is, the Nested PCR technique is a technique that prevents production of nonspecific PCR products by performing a first PCR using the outer primer pair and a second PCR using the inner primer pair. However, if the outer primer pair is not removed prior to initiating the second PCR, there may be a problem in that interaction between the outer primer pair and the inner primer pair must be considered. Furthermore, since cross contamination may occur during opening of a reaction vessel in the interim between the first PCR and the second PCR to insert PCR reactants for the second PCR to the reaction vessel, the Nested PCR technique requires a special attention.
Recently, there has been reported a fast PCR technique starting with a single molecule using a micro device in which a reaction vessel is small (E. T. Lagally et al., 2001, Anal. Chem. 73, 565˜570). In this PCR technique, however, nucleic acids may be abnormally adsorbed on a surface of the micro reaction vessel made of silicon or glass, thereby adversely affecting PCR amplification. Furthermore, PCR reactants may be evaporated due to their small volume.
Meanwhile, when PCR temperature reaches below the melting temperature of nucleic acids during the PCR, nonspecific PCR products such as primer-dimers may be produced.
To solve this problem, there have been suggested a technique in which common components of PCR amplification, such as DNA polymerase, are not inserted until a first cycle reaches the melting temperature of nucleic acids, and a hot start PCR technique. In detail, there are a method of adding common PCR components after a first cycle reaches the melting temperature of nucleic acids, a method in which a wax bead placed on a reaction solution is melted by heating to form a solidified layer having common PCR components thereon so that the reaction solution is mixed with the common PCR components after a first cycle reaches the melting temperature of nucleic acids, and a method using a Taq DNA polymerase antibody in which while a first cycle reaches the melting temperature of nucleic acids, Taq start antibody is released from Taq DNA polymerase so that the Taq DNA polymerase is activated. However, according to the above methods, there is a burden to add PCR reactants during PCR and cross contamination may occur. Furthermore, in the case of using Taq start antibody, an activation time of 10 minutes or more is normally required.
In PCR technique, the amount of PCR products after n cycles would be theoretically 2n-fold of the initial amount of target nucleic acids. The amount of PCR products in an initial PCR step increases logarithmically according to the number of cycles. However, since the annealing efficiency of primers and the synthesis efficiency of DNA double strands are actually not 100%, when the amount of PCR products reaches a predetermined level, there is observed a plateau effect in which an increase rate of PCR products decreases and amplification finally stops. In this regard, it is difficult to deduce the initial amount of target nucleic acids from the amount of PCR products. Conventionally, to quantify the initial amount of target nucleic acids, an internal standard sample is used.
Kopp et al. suggested a continuous-flow PCR on a chip in which a PCR solution flows in a reaction vessel with different temperature areas via a micro channel so that continuous PCR amplification is carried out (Martin U. Kopp et al., 1998, Science, Vol. 280, 1046˜1048). Since this PCR technique is not based on heating the entire surfaces of the reaction vessel, the reaction rate is determined by a flow rate, not a heating/cooling rate. However, separate channels for several standard samples are required for quantitative analysis, which increases a chip size. Furthermore, a large number of chips for repeated experiments are required. Amplification of different samples using a single channel may cause a problem such as contamination by DNAs adsorbed on the surface of the channel.
Baker et al. suggested PCR amplification based on fluid movement on a glass chip (Jill Baker et al., 2003, Micro TAS, 1335-1338). According to this PCR technique, the temperature of the glass chip is controlled in such a manner that the entire surface of the glass chip is raised by a heater between channels and is cooled by a cooling water beneath the glass chip. Baker et al. reported that when a fluorescent signal was measured during thermal PCR cycles in a channel filled with a diluted sample, a fluorescent peak was detected on a single molecule. However, accomplishment of this result required an analysis procedure such as removal of a background signal from weak fluorescent signal. Therefore, the analysis procedure of a fluorescent signal is obscure and it is impossible to determine whether a fluorescent peak originates from a single molecule.
Nakano et al. suggested a single molecule PCR technique using water-in-oil emulsion as a reactor (Michihiko Nakano et al., 2003, J. Biotechnology, 102, 117-124). In detail, first, an aqueous solution containing a PCR mixture, oil, and a surfactant are placed in a PCR tube and mixed using a magnetic stirrer bar to obtain the water-in-oil emulsion. Initial several cycles of PCR amplification are performed in small aqueous solution droplets. When the small aqueous solution droplets are united by phase separation of the aqueous solution and the oil by centrifugation, later cycles of PCR amplification proceed. This completes the single molecule PCR suggested by Nakano et al. Generally, when the concentration of template DNAs is very low, hybridization between primers of relatively high concentration occurs more easily, relative to that between the template DNAs and the primers, thereby producing nonspecific PCR products. In this regard, the single molecule PCR technique uses the water-in-oil emulsion as the reactor so that primary PCR amplification occurs in aqueous solution droplets containing high concentration template DNAs. When primers, which are one of reactants in the aqueous solution droplets containing the template DNAs, are completely consumed during initial several cycles of PCR, the aqueous solution droplets containing the template DNAs and another aqueous solution droplets containing no template DNAs are united by centrifugation so that secondary PCR amplification is performed by primers present in the aqueous solution droplets containing no template DNAs. However, this technique has several difficulties in actual applications. For example, use of the magnetic stirrer bar whenever preparing a water-in-oil emulsion for infectious disease diagnosis is inconvenient and increases contamination occurrence. Furthermore, the aqueous solution droplets have different sizes, which may cause a reproducibility problem. Still furthermore, in a case where the concentration of the template DNAs is very low, several repeated experiments are required. In addition, there are disadvantages in that a PCR solution has a large volume of about 50 μl and a quantitative PCR is impossible.
Meanwhile, a traditional PCR shows the qualitative results of amplified DNAs by an electrophoresis at the end-point of the PCR reaction, but has many problems such as inaccuracy of the quantitative detection of DNAs. In this regard, a Real-Time PCR was developed to allow for the quantitative detection of amplified DNAs by detecting the intensity of fluorescent light, which is in proportional to the concentration of the amplified DNAs, using an optical detection system.
However, in the case of performing the quantitative detection of amplified DNAs using a typical Real-Time PCR, there are required three or more repeated experiments using a negative control, a positive control, and at least three standard samples with different concentrations, which requires the use of a large number of reactors. It is impossible to provide such a large number of reactors for a micro PCR chip. To have such a large number of reactors, a larger-sized chip is required.